The present invention relates to a bioprocess for producing L-arginine by fermentation. The embodiments include reducing sugars obtained by enzymatic starch hydrolysis from inexpensive starch containing agro-wastes, such as Cassava bagasse and Jackfruit seed powder, both of which are abundant in Asian and African countries. The process can economically be scaled up for the production of arginine from unrefined sugar sources by replacing expensive synthetic carbon sources like dextrose or sucrose.
The growing market demand for amino acids made academia and industrialists develop new methods to produce amino acids efficiently and cost effectively. This technological race facilitated the manufacture of amino acids mainly by four methods—extraction from protein hydrolysates, chemical synthesis, enzymatic hydrolysis and fermentation. From the economic standpoint, fermentation is found to be industrially feasible and is widely used except in a few cases where high production yield has not been achieved. The economy of this method mainly depends on the cost of the carbon source, fermentation yield, purification yield and productivity in the overall process. Growth in market value for amino acids produced by coryneform bacteria led to significant developments in bioprocess and downstream processing technology. This led to the efforts to increase productivity and decrease production costs. (Thomas Hermann, “Industrial production of amino acids by coryneform bacteria,” Journal of Biotechnology 104 155-172 (2003)). Hence, any natural process that has an impact on the yield of L-arginine is in demand for utilization in industrial practice.
L-Arginine is a conditionally essential amino acid, so called, depending on the developmental stage and health status of the individual. Arginine stimulates the immune system by increasing the output of T-cells, helps in vasodilatation, in maintaining muscular health, removing ammonia from body and the release of hormones. L-Arginine has been manufactured conventionally by three methods: (i) extractions from protein hydrolysates; (ii) chemical synthesis; and (iii) enzymatic hydrolysis (Takishi Utagawa, “Production of Arginine by fermentation,” J. Nutr. 134:2854S-2857S (2004)). Later it was discovered that L-Arginine can be produced in small amounts by hydrocarbon assimilating wild strains of Corynebacterium and Brevibacterium (U.S. Pat. Nos. 3,222,258 and 3,440,141) and its mutant strains produced even higher amounts from carbohydrates (British Patent No. 1,278,917).
Among regulatory mutants of various micro organisms, Corynebacterium glutamicum showed higher production of L-arginine (Hajime Yoshida, Kazumi Araki and Kiyoshi Nakayama, “Arginine Production by Arginine Analog-resistant Mutants of Microorganisms,” Agric. Biol. Chem., 45 (4), 959-963 (1981). Some of the most typical arginine producing mutants are of the genus Corynebacterium resistant to 2-thiazolealanine (U.S. Pat. Nos. 3,723,249 and 3,878,044) and canavanine (U.S. Pat. No. 3,849,250; UK Patent No. 1,351,518). Mutants of the genus Bacillus (U.S. Pat. Nos. 3,734,829 and 4,086,137, 4,430,430) and Escherichia (U.S. Pat. Nos. 4,430,430, 6,897,048) also are also found to produce Arginine in substantial amounts.
The production of L-Arginine in a fair amount by microbial fermentation was first reported by Kisumi et al, “Production of L-Arginine by Arginine Hydroxamate-Resistant Mutants of Bacillus subtilis,” Appl. Microbiol. 22, 987 (1971). Oxygen supply is known to have an important influence on aerobic amino acids production by microorganisms (Takishi Utagawa, “Production of Arginine by fermentation,” J. Nutr. 134:2854S-2857S (2004)). Growth under anaerobic condition often leads to formation of toxic by-products such as acetic acid and ethanol, which in turn strongly inhibit L-arginine production (J. Gong, J. Ding, H. Huang, Q. Chen, Kinetic study and modeling on L-arginine fermentation. Chin. J. Biotech. 9 (1) 9-18 (1993).)
Cassava (Manihot esculents) bagasse has been used for the production of L-(+)-lactic acid by Lactobacillus casei and Lactobacillus delbrueckii. (Rojan P. John, K. Madhavan Nampoothiri and Ashok Pandey, Applied Biochemistry and Biotechnology, Vol 34, p 263-272 (2006); Rojan P. John, K. Madhavan Nampoothiri and Ashok Pandey, “Solid state fermentation for L-lactic acid production from agro wastes using Lactobacillus delbrueckii,” Process Biochemistry, Vol. 41 p:759-763 (2006). It was also used in production of giberellic acid (A. Tomasini l, C. Fajardo and J. Barrios-Gonza'lez, “Gibberellic acid production using different solid-state fermentation systems,” Vol 13. p 203-206 (1997)) and citric acid by SSF (Flavera Camargo Prado, Luciana Porto de Souza Vandenberghe and Carlos Ricardo Soccol, “Relation between Citric Acid Production by Solid-State Fermentation from Cassava Bagasse and Respiration of Aspergillus niger LPB Semi-Pilot Scale,” Brazilian archives of Biology and Technology, Vol. 48, Special n.: pp. 29-36 (2005)). Recently, Ubaid et al reported the use for gamma linolenic acid production (Syed Ubaid et al., “Enrichment of γ-linolenic acid in the lipd extracted from Mucor zychae MTCC5420,” Food Research International, Volume 42, issue 4, May 2009, Pages 449-453).
Similarly, Jack fruit seed powder has been used in the production of pigments (Sumathy Babitha, Carlos R. Soccol and Ashok Pandey, “Jackfruit Seed—A Novel Substrate for the Production of Monascus Pigments through Solid-State Fermentation,” Food Technol. Biotechnol, 44 (4) 465-471 (2006)) and in production of Poly hydroxybutyrate (Ramadas NV, Soccol CR, Pandey A Appl Biochem Biotechnol, “A Statistical Approach for Optimization of Polyhydroxybutyrate Production by Bacillus sphaericus NCIM 5149 under Submerged Fermentation Using Central Composite Design” Appl. Biochem. Biotechnol. (2009)).
Some of the disadvantages of the existing fermentation process of Arginine include, for example, the lack of wild type cultures capable of arginine production unlike in the case of glutamate, where wild C. glutamicum is capable of producing large amounts. It also has been difficult to produce auxotrophic mutants capable of arginine production, and there is a need for genetic engineering for strain improvement suited to arginine production. In addition, the production costs of producing arginine are relatively high due to the use of pure glucose as the sole carbon source. Hence, inexpensive alternatives to enhance the yield using the available strains are in desirable.